The present invention generally relates to a process for the preparation of hydroxyalkyl starch, and more particularly to the preparation of hydroxyethyl starch having utility as a cryoprotective agent for human erythrocytes.
Hydroxyethyl starch (HES), prepared by the reaction of starch with ethylene oxide, is readily water soluble and, in contrast to native starch, aqueous solutions are stable over prolonged periods. These properties, together with the low cost of manufacture have led to its extensive use as an industrial chemical.
In commerical practice, hydroxyalkyl starch is prepared by the reaction of alkylene oxides with starch, often in the presence of catalysts such as inorganic salts, inorganic alkalis, and certain organic bases. Reaction systems have been designed to produce an essentially granular product by inhibition of gelation, by limiting the amount of water present in the reaction system, by the addition of inorganic salts, or by carrying out the reaction in certain alcoholic media. Many of these methods are disclosed in U.S. Pat. Nos. 2,516,632, 2,516,633, 2,516,634 and 2,845,417 to Kesler and Hjermstad.
In the last decade considerable interest has been shown in the pharmacological properties of HES as a blood plasma volume expander. See, for example, U.S. Pat. No. 3,523,938.
More recently, research has been undertaken to develop cryoprotective agents suitable for use in processes for the long-term, large volume storage of blood through freezing. It has been found that to prevent extensive haemolysis of the blood cells during the freezing and thawing procedures, it is necessary to mix the erythrocytes with a cryoprotective agent. Dimethyl sulphoxide and glycerol have been proven to be effective cryoprotective agents. They act by an intracellular mechanism so that when the erythrocytes are thawed and prepared for transfusion into a patient, it is necessary to carry out a treatment of the cells which removes these protective agents from within the cells.
Polyvinyl pyrrolidone and hydroxyethyl starch (HES) have also-been investigated as cryoprotective agents. Both of these substances work by an extracellular mechanism and hydroxyethyl starch has the advantage that it can be transfused, if desired, together with the erythrocytes so that no separation of the cryoprotective agent from the cells is necessary before use. See, for example, U.S. Pat. No. 3,758,382.
The hydroxyalkyl starch used as a plasma volume extender and as a cryoprotective agent for human erythrocytes is prepared from a so-called waxy starch. The reason for the choice of a hydroxyalkyl derivative of waxy starch as an artificial colloidal agent is that waxy starch is made up primarily of amylopectin and little or no amylose. Amylopectin, the branched component of starch, is very similar in structure to glycogen, the reserve polysaccharide of animals. Due to this similarity it was concluded that a hydroxyalkyl derivative of amylopectin should be compatible with body tissue, and more resistant to the highly-specific enzyme systems which attack it. Waxy starch can be obtained from genetic varieties of maize, sorghum, rice and barley.
One process which has been developed for the production of HES which is suitable as a cryoprotective agent for human erythrocytes is described by T. J. Schoch in the paper "Preparation and Characterization of Hydroxyethyl Starch" in the Proceedings of the Third Conference on Artificial Colloidal Agents, National Academy of Sciences, National Research Council, Washington, D.C., (1965).
The process comprises:
i. a preliminary alkali treatment with aqueous sodium hydroxide to reduce the content of protein and natural pigments in the granular starting material,
ii. acid hydrolysis with dilute hydrochloric acid to reduce the molecular size of the starch so that an aqueous solution of the final product has the desired viscosity and osmotic pressure.
iii. dissolution of the hydrolyzed starch granules in aqueous sodium hydroxide,
iv. treatment of the resulting solution with ethylene oxide which reacts with free hydroxyl groups of the hydrolysed starch to form hydroxyethyl groups (without this partial conversion of some of the free hydroxyl groups, the hydrolyzed starch would be too readily broken down by blood amylase and would be excreted from the body before their full effect as a blood volume extender was utilized), the molar substitution, i.e. the numbers of hydroxyethyl ether groups per anhydroglucose unit in the starch, being from 0.7 to 0.9,
v. carbon treatment to remove residual colour,
vi. filtration to remove haze and particulate material,
vii. spray-drying to give a dry white product,
viii. acetone extraction to remove by-product glycols formed in the hydroxyethylation step, and
ix. drying to remove acetone.
It can be seen that the process is lengthy and, when operated on a commercial scale, requires a considerable amount of equipment. The product obtained also suffers from the disadvantage that it contains a significant amount of inorganic salts as an impurity. Figures of 2.84 and 2.75% for the ash content, calculated as NaCl, are quoted in the Schoch paper referred to above and figures of 1.20 and 2.03% NaCl are quoted by a commercial supplier who is believed to produce HES according to the process described in the Schoch paper.
The presence of such a proportion of inorganic salts in hydroxyethyl starch is disadvantageous because it precludes the use of reversible agglomeration as a method of post-thaw processing of human erythrocytes when the hydroxyethyl starch is used as a cryoprotective agent.